The goal of this career development award is to integrate the genetics of autism spectrum disorders (ASD), developmental neuroscience, and the functional analysis of rare variants, in order to advance our understanding of the basic biological mechanisms underlying ASD. Dr. Hoffman is a board certified, practicing child psychiatrist, who is currently pursuing her PhD in Investigative Medicine in the laboratory of Matthew State, MD, PhD, at the Yale Child Study Center. Her career goal is to become an independent investigator with dual expertise in the genetics of child psychiatric disorders and the neurobiology of vertebrate systems. In this application, she proposes to utilize zebrafish as a novel translational tool that will leverage findings from human genetic studies as a means of advancing our knowledge of the pathophysiology of ASD. Dr. Hoffman will gain this expertise through the combined guidance of her primary mentor, Dr. State, a leader in ASD genetics, and co-mentor, Antonio Giraldez, PhD, an expert in zebrafish development, which will promote the establishment of this innovative approach to investigating the role of ASD susceptibility genes in neural circuit formation. The objective of this research is to elaborate basic mechanisms of ASD by investigating the function of the ASD risk gene, Contactin Associated Protein-2 (CNTNAP2) in neural development, and to determine how sequence variants in this gene identified in individuals with ASD disrupt its function. Using the emerging technology of zinc finger nucleases, which have superior accuracy over morpholinos, Dr. Hoffman induced targeted germline mutations in the two zebrafish CNTNAP2 genes, CNTNAP2a and 2b. To our knowledge, this is the first zebrafish knockout of an ASD risk gene generated by this method. Our hypothesis is that CNTNAP2a/2b double knockouts will display reproducible morphological and/or behavioral phenotypes that will yield important insights into the function of CNTNAP2 in neural development. This hypothesis will be tested by pursuing these aims: 1) Generate double knockouts of the two zebrafish CNTNAP2 genes by crossing fish carrying germline mutations; 2) Identify quantifiable morphological and/or behavioral phenotypes in CNTNAP2 double knockouts; and 3) Characterize the ability of the human CNTNAP2 gene with rare sequence variants found in individuals with ASD to reverse the phenotypes. Our rationale for this approach is that the development of an in vivo system to rapidly assess the functional consequences of rare genetic variants is the crucial next step in understanding the biology of ASD. Dr. Hoffman has assembled an outstanding team of mentors and collaborators, including pioneers in zebrafish neural circuit analysis and behavioral phenotyping, as this system is anticipated to provide unique insights into the role of CNTNAP2 in the neural circuitry underlying simple behaviors. The extensive resources of the Yale Child Study Center, together with her formal plan for didactics in neuroscience and developmental biology, will further support Dr. Hoffmans goal of elucidating the molecular and cellular mechanisms of ASD.